Structural complexity of Dengue virus untranslated regions: cis-acting RNA motifs and pseudoknot interactions modulating functionality of the viral genome

Sztuba-Solinska, Joanna; Teramoto, Tadahisa; Rausch, Jason W.; Shapiro, Bruce A.; Padmanabhan, Radhakrishnan; Le Grice, Stuart F. J.

Probing analysis of DENV-MINI RNA verifies the 5 0 -3 0 -UTRs long-range interactions and suggests a TL1/ PK2 pseudoknot SHAPE has been developed to study RNA secondary structure by examining backbone flexibility (directly related to the base pairing) at each nucleotide position via reactivity with a specific electrophilic reagent (21) . We applied this technique to the in vitro-synthesized 719-nt subgenomic 'minigenome' RNA (DENV-MINI) template, which contains the 5 0 -terminal 226 nt, 42 nt from the C-terminal coding region of NS5, including the UAG termination codon, and the 451-nt 3 0 terminus (18) . Previous studies showed that this RNA molecule harbored essential cis-acting elements for efficient translation (25, 26) , as well as (À) strand RNA synthesis in vitro by either DENV2-infected cell lysates or purified RNAdependent RNA polymerase (RdRP) (27, 28) . DENV-MINI RNA was also used for secondary structure prediction analysis using the MPGAfold algorithm (29) to infer the structure-function role of the TLs and PKs regions in DENV replication and translation (8) .